Particulate analyte characterization is currently limited to averaging techniques that sample a field of particles under comparatively mild sampling conditions, or single particle interrogation techniques that subject the particle to a harsh sampling condition in order to generate a sufficient signal relative to background noise. Unfortunately, many forms of particulate, including organic polymers and biological cells cannot tolerate the spectroscopic techniques needed to characterize them directly. Instead, one resorts to staining or shadowing techniques that are both labor intensive and destructive of the test subject. The time required to characterize particulate also limits the use of such information in rapidly changing situations as diverse as a manufacturing, treating an infection, and managing a contamination event.
Accurate and sensitive detection of viruses and hazardous microorganisms is a requirement in numerous industrial, military, workplace, healthcare and even home environments. A difficulty that arises in biological detection is the relative paucity of molecules that are exclusively found in targets. Additionally, the inability of a single technique to afford definitive characterization through the strain level of the microbe means that culturing the sample is often required thereby increasing the time-consumed in characterization. Animate particulate, such as that found in a forensic setting, simply is not fully characterized.
Since the seminal work of Ashkin in 1970, based on a dual laser beam system (1) and later work in 1986 employing a single laser beam apparatus (2), the laser tweezer phenomenon has been broadly accepted as a powerful tool to study viruses (3), vegetative bacterial cells (4-8), mammalian cells (9) and colloidal crystallization in microgravity environments (10). More recently, this technique in combination with various Raman detection schemes has been applied to the investigation of inorganic gas bubbles (11), aerosols (12), emulsion particle polymerization (13), liquid-liquid extraction of toluene in water (14), organic nanoparticles (15), yeast cells (8) and solid-phase peptide synthesis (16).
Raman spectroscopy has been an invaluable technique in the study of various chemical systems and has become widely accepted as an analytical characterization methodology (17,18). The attractiveness of this technique stems from the attributes of narrow spectral band structure, lack of interference from water, and relative insensitivity to the excitation wavelength employed. However, un-enhanced Raman spectral features are considered to be relatively weak thus requiring relatively lengthy collection times. In many applications, high quality spectra may be achieved with shortened acquisition times and improved spectral features by exploiting various amplification techniques. Namely, Resonance Raman Scattering (RRS), Surface Enhanced Resonance Raman Scattering (SERRS) or Surface-Enhanced-Raman-Scattering (SERS) (17-19). RRS and SERRS are typically conducted using ultraviolet excitation to facilitate electronic excitation (i.e. strong absorption) in the analyte of interest. Ultraviolet excitation precludes implementation of RRS and SERRS in a single beam optical trapping configuration since the trapped particle experiences MW/cm2 UV intensity levels that coupled with significant UV-light absorption photo-decompose most analytes.
SERS represents another method for enhancing a conventional Raman signal that is operative with less destructive near-infrared optical beam trapping. Currently, the mechanism of the SERS effect is not fully understood; however, a plausible explanation for at least a significant portion of the spectral amplification has been attributed to an increase in the electromagnetic field strength encountered by the analyte. Briefly, this intensified electromagnetic field is generated when a roughened metal surface (typically, Au, Ag, Pt, Pd or Cu) is irradiated with the requisite wavelength of light and metal conduction band electrons are excited to collective oscillation and produce a surface plasmon resonance. The Raman and infrared (IR) signals associated with analytes proximal to such a metal surface have been shown to be enhanced from 4 to 14 orders magnitude relative to un-enhanced Raman (17). Additionally, the enhancement is reversible when the particulate analyte is removed from the metal surface. In spite of these features, SERS has gained only limited acceptance since early reports on this technique in the late 1970's. This limited acceptance is in large part due to the lack of fabrication reproducibility in most SERS substrates. Relatively recent advances in SERS-active substrate fabrication have addressed this reproducibility issue. Specifically, these substrates are easily wavelength adjustable, durable, biocompatible and possess a long shelf-life (19).
Bacterial spore characterization is a subset of particulate characterization where the limitations of current techniques are profound. Endospores are an alternate cell form of various bacterial genera including Bacillus, Clostridium, Desulfotomaculum, Oscillospira, Sporosarcina, Sporolactobacillus, and Thermoactinomyces. An endospore is formed as an adaptive response to environmental conditions unfavorable to the bacterial cell form, such as dehydration, limited nutrient availability and extreme temperatures. While endospores are themselves metabolically inactive, they are activated under appropriate conditions, forming an active vegetative bacterial cell. Thus, the presence of an endospore is indicative of a potential biohazard and therefore endospore detection is a desirable goal.
Over the past three decades Raman spectroscopy has been employed to study various Bacillus spore species. Due to the relatively meager Raman scattering cross-sections exhibited by many biological materials, a majority of this reported work has been conducted using various Raman amplification techniques. Specifically, early work in this area was based on the Resonance-Raman-Scattering (RRS) technique utilizing ultraviolet (i.e., 222 nanometer) to green (i.e., 514.5 nanometer) laser excitation sources (20-23). More recently, reports have been published describing the implementation of Femtosecond Adaptive Spectroscopic Techniques applied to Coherent-Anti-stokes-Raman-Spectroscopy (FAST CARS) (24) as well as SERS microscopy (25). These various Raman spectroscopies have been applied to the study of bulk endospore samples and have failed to afford information sufficient to distinguish between strains of the same endospore.
Thus, there exists a need for a non-destructive characterization technique for single particles. The ability to characterize a single particle in a non-destructive manner confers the ability to examine particulate changes over time and to assess population diversity.